The gene bifocal (bif), required for photoreceptor morphogenesis in the Drosophila compound eye, encodes a protein that is shown to interact with Protein phosphatase 1 (PP1: PP1-87B) using the yeast two-hybrid system. Complex formation between Bif and PP1 is supported by coprecipitation of the two proteins. Residues 992 to 995 (RVQF) in the carboxy-terminal region of Bif, which conform to the consensus PP1-binding motif, are shown to be essential for the interaction of Bif with PP1. The interaction of PP1 with bacterially expressed and endogenous Bif can be disrupted by a synthetic peptide known to block interaction of other regulatory subunits with PP1. Null bif mutants exhibit a rough eye phenotype, disorganized rhabdomeres (light-gathering rhodopsin-rich microvillar membrane structures in the photoreceptor cells) and alterations in the actin cytoskeleton. Expression of wild-type bif transgenes results in significant rescue of these abnormalities. In contrast, expression of transgenes encoding the Bif F995A mutant, which disrupts binding to PP1, was unable to rescue any aspect of the bif phenotype. The results indicate that the PP1-Bif interaction is critical for the rescue and that a major function of Bif is to target PP1c to a specific subcellular location (Helps, 2001).
The studies presented here suggest that a major function of Bif is to target PP1c to a specific subcellular location to regulate the normal developmental pattern of the eye. At the molecular level, the organization of the actin cytoskeleton is dependent on the Bif-PP1 interaction, suggesting that PP1 may influence actin movement or operate in a pathway that regulates actin distribution within the cell. Although the actin cytoskeleton is a highly ordered structure, it is very dynamic, undergoing changes that affect cell shape, motility, and adhesion. Bif does not possess a known actin-binding motif, but its subcellular location within the eye is consistent with its playing a role in actin function and possibly binding to some component of the actin cytoskeleton. Recently two novel actin-binding proteins found in mammalian neurons, neurabin I and II, have been shown to bind PP1c. Neurabin I was highly concentrated at the synapse of developed neurons and in the lamellopodia of the growth cone during the development of neurons, suggesting that it is required in synapse function and formation. Suppression of endogenous neurabin I expression with antisense oligonucleotides in hippocampal neurons inhibits neurite outgrowth. Neurabin II is ubiquitously expressed but is enriched in the postsynaptic density fraction of the brain. The presence of a PDZ domain that might bind to a transmembrane protein and their localization make it likely that neurabins I and II bind at the plasma membrane. The neurabins have therefore been suggested to serve as linkers between the actin cytoskeleton and the plasma membrane at cadherin-based cell-cell adhesion sites and to localize PP1c to the plasma membrane in dendritic spines of neurons, where the complexes may modulate synaptic transmission. Both neurabins I and II show F-actin cross-linking activity, as do the -actinin-spectrin family of actin-binding proteins. However, the actin-binding sites on the neurabins are distinct from other known actin-binding sites. Bif does not appear to be a Drosophila homolog of the mammalian neurabins, because it has no sequence similarity to these proteins and its tissue localization is distinct, In addition, there is a gene (CG16757) located at 62E6-8 in the Drosophila genome that encodes a putative neurabin homolog. However, the Bif-PP1c complex may serve functions in the photoreceptor cells of the eye analogous to those of the neurabin-PP1c complexes in other tissues, transmitting or modulating signals, possibly from cell-cell contacts, which cause a rearrangement of the actin cytoskeleton. This suggests that PP1c may play a general role in modulating the actin cytoskeleton (Helps, 2001 and references therein).
Bif inhibits the phosphorylase phosphatase activity of PP1c, similarly to a number of other PP1c-binding proteins, such as the myosin binding subunits and 53BP2. Neurabins I and II also inhibit the phosphorylase phosphatase activity of PP1c. Neurabin I has been shown to be phosphorylated in vitro by protein kinase A (PKA), which decreases its binding to PP1c. In addition, mutation of the phosphorylatable serine to glutamic acid reduces the inhibitory activity of neurabin, suggesting that the complex participates in a cyclic AMP-PKA signaling mechanism. Bif has several potential Ser/Thr phosphorylation sites; in the vicinity of the PP1-binding motif, the carboxy-terminal sequence of Bif, RRSSTIM, could serve as a potential phosphorylation site for PKA (as well as for a number of other kinases). Phosphorylation of Bif could affect PP1c binding and/or activity, allowing the Bif-PP1c complex to modulate signaling processes. Alternately or in addition, the Bif-PP1c complex might be required to dephosphorylate proteins associated with actin. Actin-binding proteins can bind to actin monomers, cross-link actin filaments into bundles or gels, sever actin filaments, or cap their growing ends. Some, such as myosin II and cofilin, are known to undergo phosphorylation. Dephosphorylation of such proteins, possibly by PP1c complexes, may effect a redistribution of the actin cytoskeleton (Helps, 2001 and references therein).
The genetic interaction between misshapen (msn) and bif may reflect a direct physical interaction between Msn and Bif. Alternatively, Msn and Bif may interact indirectly through additional proteins. To distinguish among these possibilities, tests were performed to see if Bif physically interacts with Msn. Recombinant proteins containing fragments of Bif were generated. Interestingly, it was found that immobilized glutathione-S-transferase (GST) fusion protein containing the Bif carboxy-terminal fragment (amino acids 6491063) (GST-C-Bif) can precipitate Msn in a dosage-dependent manner from cell lysates. The immobilized GST-C-Bif is also able to precipitate Msn from solutions containing only affinity-purified Msn, indicating a direct interaction between Msn and Bif. To determine if Msn and Bif also form a complex in vivo, COS-7 cells were cotransfected transiently with Msn and epitope-tagged Bif expression constructs. The formation of complexes was examined by coprecipitation. Msn copurifies with Bif from cell lysates, indicating an in vivo association of Msn with Bif (Ruan, 2002).
To further determine if Bif is a substrate of Msn, an in vitro kinase assay was performed. Recombinant Msn protein was prepared using the Drosophila expression system. Msn strongly phosphorylates recombinant Bif protein containing either the carboxy-terminal fragment (i.e., GST-C-Bif) or an amino-terminal fragment (amino acids 1563) of Bif. In summary, these experiments indicate that Msn is capable of associating directly with Bif and phosphorylating Bif (Ruan, 2002).
Some GCKs can activate downstream kinases independently of their kinase domains. While the intrinsic kinase activity of Msn has been shown to be required for regulating dorsal closure during early embryonic development, it was of interest to enquire if the role of Msn in R cell growth cones is also dependent on its kinase activity. To address this, a test was performed to see if a kinase-defective form of Msn (Msn D160N) could rescue R cell projection defects in msn mutants. Wild-type and mutant UAS-msn were expressed under control of the neuronal-specific elav-Gal4 driver in larvae that are homozygous for a msn hypomorphic mutation msnl(3)03349. While most msnl(3)03349 embryos eventually develop into pupae, they all die at the late pupal stage. msnl(3)03349 third-instar larvae display a mild defect in R cell axonal projections. Neuronal-specific expression of wild-type Msn rescues the R cell projection defect in all individuals examined. In contrast, no rescue was observed with the kinase-defective Msn (D160N) in all hemispheres examined. Similarly, Msn (D160N) is unable to induce premature growth cone termination when overexpressed in R cells under control of the GMR eye-specific promoter. Neuronal-specific expression of Msn (D160N) also decreases the viability of msn mutant embryos; only 5% of msnl(3)03349 embryos expressing Msn (D160N) survived to the pupal stage (compared to >90% in the absence of this transgene), suggesting that Msn (D160N) functions as a dominant-negative form to inhibit the function of either maternal msn or residual zygotic msn or both in early msnl(3)03349 embryos (Ruan, 2002).
While studies indicate the involvement of Bif in the control of growth cone cytoskeleton, the exact role of Bif in regulating cytoskeletal changes is unclear. To address this, the effect of Bif expression on the level and the distribution of F-actin was examined in COS-7 cells. Untransfected cells show a low level of F-actin, which is mainly found at cell periphery and is occasionally present as thin fibers within the cell body. Expression of Msn alone affects neither the level of F-actin nor cell morphology. To examine the effect of Bif on actin cytoskeleton, COS-7 cells were transiently transfected with a construct encoding a GFP-Bif fusion protein. Remarkably, it was found that GFP-Bif not only colocalizes with F-actin in transfected cells, it also greatly increases the level of F-actin and induces striking morphological changes. Many Bif-positive cells formed a large number (>20) of microspikes (<5 µm) and long thin projections that resemble filopodia (>5 µm). Filopodium formation was correlated with the level of GFP-Bif expression, since all cells displaying multiple filopodia-like structures expressed high level of GFP-Bif (Ruan, 2002).
To determine the effect of Msn on Bif-induced cytoskeletal changes, COS-7 cells were cotransfected with Msn and GFP-Bif constructs. While the level of F-actin in cells coexpressing Msn and GFP-Bif remained high, similar to that in cells expressing GFP-Bif alone, the organization of F-actin was significantly altered. In cells expressing GFP-Bif alone, actin tended to form long fine fibers. In contrast, in cells coexpressing Bif and Msn, actin fibers were frequently seen as short large bundles and irregular large aggregates. The number of cells with filopodia-like structures also decreased significantly when cotransfected with Bif and Msn. Filopodia in cells coexpressing Bif and Msn were much shorter and less branched than that in cells expressing Bif alone. Only ~13% filopodia measured in cells coexpressing Bif and Msn were >10 µm in length, while ~47% filopodia measured in cells expressing Bif alone were >10 µm in length. To determine if the intrinsic kinase activity of Msn is required for its effect, the kinase-defective form of Msn (D160N) was also included in the experiments. Unlike Msn, Msn (D160N) affects neither the organization of F-actin nor the formation of filopodium in Bif-positive cells (Ruan, 2002).
In summary, Bif promotes actin polymerization and induces filopodium formation in cultured cells. Although Msn alone does not affect F-actin nor cell morphology, it modulates the effect of Bif on actin cytoskeleton. The action of Msn is dependent on its intrinsic kinase activity (Ruan, 2002).
Transport, translation, and anchoring of osk mRNA and proteins are essential for posterior patterning of Drosophila embryos. Homer and Bifocal act redundantly to promote posterior anchoring of the osk gene products. Disruption of actin microfilaments, which causes delocalization of Bifocal but not Homer from the oocyte cortex, severely disrupts anchoring of osk gene products only when Homer (not Bifocal) is absent. The data suggest that two processes, one requiring Bifocal and an intact F-actin cytoskeleton and a second requiring Homer but independent of intact F-actin, may act redundantly to mediate posterior anchoring of the osk gene products (Babu, 2004).
Both Bif and Hom show asymmetric localization at the apical cortex of embryonic neuroblasts, indicating that these F-actin binding proteins may be involved in neuroblast asymmetric divisions. However, animals lacking both the maternal and zygotic components of either gene are fertile, viable, and show no obvious defects in embryonic CNS development. This prompted construction of double mutants of bif and hom. However, although double homozygous mutant females are viable, they show defects in oogenesis, with the vast majority of the eggs produced remaining unfertilized, as judged by the lack of staining in eggs using an antibody directed against the sperm tail. In the few fertilized embryos that do undergo development, the numbers of Vasa positive germ cells are drastically reduced, suggesting possible defects in the function or localization of posterior determinants during oogenesis (Babu, 2004).
Analyses of bif and hom single mutants as well as double mutant oocytes indicate that the two genes act in a redundant manner for the correct anchoring of posteriorly but not anteriorly localized molecules. In stage 10 oocytes, posterior group molecules, including oskar (osk) RNA, the two isoforms of Osk proteins, Staufen (Stau), and a fusion protein in which ß-galactosidase has been fused to the N-terminal extension of the long form of the Osk protein (referred to as Osk-ßGal, used as a marker for the long form of the Osk protein, which has been shown to have a role in the posterior cortical maintenance of Osk), are all localized as tight posterior cortical crescents in wild-type oocytes. In most double mutant oocytes, these molecules, when detectable, are present largely at the posterior region; however in contrast to wild-type oocytes, they show a diffuse distribution that extends into regions of the posterior cytoplasm distinctly interior to the posterior cortex. In about 30% of the cases, Osk or Stau protein cannot be detected. The defects seen in the oocytes of double mutants are essentially absent in the single mutant oocytes. These findings indicate that whereas bif and hom are individually dispensable, together they are required for the localization of the posterior components of the oocytes. These defects in localization are specific for the posterior group molecules, because the anterior/dorsal localization of Gurken and anterior localization of bicoid RNA are unaffected (Babu, 2004).
Not surprisingly, staining with anti-Bif and anti-Hom antibodies indicates that both proteins are expressed in the oocyte. Bif localizes to the oocyte cortex in a manner very similar to that seen for F-actin. The staining seen with the anti-Hom antibody is highly punctated and, although localization is cortically enriched, Hom is also present in the cytoplasm. The cortical staining seen in wild-type oocytes (and nurse cells) is absent in mutant oocytes stained with the corresponding antibodies, confirming the specificity of both antibodies and that the mutant alleles do not produce detectable amounts of protein. Since homLL17 is a complete deletion of the coding region and bifR47 removes a significant portion of the coding region, they are both likely to be null alleles (Babu, 2004).
Several observations indicate that the defect in posterior localization of the osk gene products is due to defective anchoring and not transport or translation of osk RNA. Osk RNA and Osk proteins as well as Stau are localized normally in stage 9 double mutant oocytes. Consistent with this, both the F-actin and microtubule cytoskeletons in the double mutants are indistinguishable from those in the wild-type oocytes. Not only does the polarity of the microtubules appear normal, as assayed using a kinesin heavy chain (khc) ß-Gal marker, the cytoskeleton-dependent cytoplasmic streaming is also absent in stage 9 oocytes and occurs normally in stage 10 double mutant oocytes, as is seen with wild-type oocytes. Taken together, these observations indicate that the double mutant oocytes retain, at a gross level, normal cytoskeletal structure. They can transport osk mRNA to the posterior cortex, and translate it appropriately, but do not maintain the posterior anchoring of the osk gene products (Babu, 2004).
An intact F-actin cytoskeleton is thought to be required for asymmetric protein localization in several contexts. In the oocyte, loss or reduction of the actin binding proteins moesin and tropomyosin have been shown to affect the posterior anchoring of Osk in the oocyte and the embryo, respectively. To assess the requirement for an intact microfilament cytoskeleton for the anchoring of osk RNA and proteins in the oocyte, the localization of these molecules was examined in wild-type oocytes treated with an actin depolymerizing drug, Latrunculin A (Lat A). Following treatment with 20 µm Lat A, cortical F-actin in the oocytes was largely undetectable with phalloidin, yet, unexpectedly, both Osk proteins (short and Osk-ßGal) and osk RNA remain localized to the posterior cortex of the great majority of wild-type oocytes from stage 9-10B. This was seen even when the oocytes were overstained for the osk RNA: in around 17% of the Lat A-treated wild-type oocytes, mild defects in anchoring are observed. osk RNA and proteins show a diffuse localization at the posterior cortex. However, in no cases were they seen concentrated in the cytoplasm or had they become delocalized or undetectable (as seen in the hom/bif double mutant oocytes) as would be expected if an intact F-actin cytoskeleton were to be an absolute requirement for the normal anchoring of Osk. These mild effects on protein localization in the oocyte are in distinct contrast to those seen in embryonic neuroblasts where severe and high-penetrance defects in asymmetric protein localization are observed following disruption of microfilaments. These observations suggest that the role of microfilaments in the anchoring of proteins to the cortex may differ in the different cellular contexts (Babu, 2004).
Additional experiments were performed to ascertain whether the mild effects on Osk posterior anchoring following disruption of microfilaments are peculiar to Lat A treatment. In fact, the posterior cortical localization of Osk remained in the great majority of oocytes even after treatment with cytochalasin D (CD), Lat A followed by CD, CD followed by Lat A, and up to 100 µM Lat A. These results are consistent with previous reports of CD disruption of F-actin, for example. However, they indicate that an intact F-actin cytoskeleton, although required for the normal posterior anchoring of Osk in a small proportion of oocytes, is probably not the only factor involved in normal anchoring of Osk to the posterior cortex. There are at least two possible explanations for these observations. First, a small amount of residual F-actin might remain even after sequential treatment with Lat A and CD, which is sufficient to anchor Osk normally in a small fraction of the drug-treated oocytes. Alternatively, there may be other factors besides an intact F-actin cytoskeleton, which can, in parallel, contribute toward the posterior anchoring of osk RNA and proteins (Babu, 2004).
The requirement for intact microfilaments on Hom and Bif localization was assessed. Both Bif and Hom localize to the cortex (and in the case of Hom also the cytoplasm) of wild-type oocytes. Following depolymerization of F-actin with Lat A, such that cortical F-actin becomes undetectable in the oocyte, Hom localization appears unchanged from the wild-type pattern in all oocytes, but Bif becomes highly diffuse with essentially no detectable enrichment at the cortex. This appears to be an effect on Bif localization and not stability, because the levels of the protein are not reduced as judged by Western blot analysis. Treating oocytes with colchicine, which disrupts the microtubules, did not affect either Hom or Bif localization in the oocyte. These findings raise the possibility that bif function might be dependent on intact F-actin; however, Hom localization is Lat A-insensitive, suggesting that its function in the oocyte may not require intact microfilaments. However, the possibility cannot be excluded that Lat A treatment allows for the retention of a small amount of the F-actin cytoskeleton, and this is stabilized in some way by Hom (Babu, 2004).
These data raise the possibility that there might be two processes, one that is microfilament-dependent and requires Bif, and another which is not dependent on intact microfilaments and requires Hom. Either process is sufficient to anchor the osk gene products to the posterior cortex of the great majority of the oocytes. One prediction of this hypothesis is that hom should be necessary to anchor posterior components in the absence of intact F-actin. Indeed, when hom single mutant oocytes were treated with Lat A, a large amount of cytoplasmic Osk was found at stage 9 and 10 near the posterior pole, and there was a large reduction in the Osk-ßGal signal. This could indicate that the loss of the longer Osk isoform may be the primary defect seen in Lat A-treated hom mutants; this longer Osk isoform is known to be essential for osk RNA and protein anchoring. The defects induced by depolymerizing F-actin in hom oocytes are similar to but more severe than those seen in bif;hom double mutant oocytes. This is probably due to the fact that F-actin disruption also leads to premature streaming in stage 9 and enhanced streaming in stage 10 oocytes, thus accentuating the effects of the loss of Osk anchoring at the posterior cortex (Babu, 2004).
The above results demonstrate that disruption of F-actin in the absence of hom function disrupts anchoring of the osk gene products. Similar results were obtained when hom mutants were treated with just CD or treated successively with Lat A and CD or vice versa. CD does not cause loss of F-actin as seen with phalloidin staining, and causes changes in the cortical F-actin as well as causing some of the F-actin to be seen in the cytoplasm. This latter effect is not seen with Lat A. This could be attributed to the difference in the mechanism of action between CD and Lat A. However, despite this difference between CD and Lat A, the effects of these drugs singly or in combination on Osk posterior anchoring are similar, causing mild defects in Osk posterior anchoring in only one-fifth of the treated wild-type oocytes and severe defects in the great majority of treated hom oocytes (Babu, 2004).
A second prediction is that disruption of microfilaments in the absence of bif should not affect anchoring of Osk. Indeed, most wild-type and bif single mutant oocytes treated with Lat A show largely wild-type anchoring of the Osk gene products, similar to Lat A treatment of wild-type oocytes. These results are consistent with the notion that Bif functions in an F-actin-dependent manner in maintaining Osk to the posterior of the oocyte (Babu, 2004).
If there are two independent mechanisms that act redundantly for normal Osk anchoring at the posterior cortex, then it would follow that the bif;hom double mutants in the absence of an intact F-actin cytoskeleton would show a phenotype similar to that of hom single mutants treated with Lat A, and not a more severe phenotype. This is indeed the case that is observed on testing osk RNA and Osk proteins in Lat A-treated double mutant oocytes (Babu, 2004).
In light of the finding that Hom posterior cortical localization remains unchanged following F-actin disruption, its ability to localize Osk to the posterior may be because it forms a complex with Osk. Co-immunoprecipitation experiments, using Drosophila ovarian extracts, indicate that Hom and Osk form a complex in vivo. Further, the stability of this complex is not dependent on an intact F-actin cytoskeleton (Babu, 2004).
The maintenance of Osk at the posterior of the oocyte may be mediated by two distinct mechanisms, either of which is sufficient, at least for the great majority of the oocytes. One mechanism does not require an intact F-actin cytoskeleton, and Hom seems to be an important player in this process. Hom can complex with Osk, and the stability of this complex is not dependent on an intact F-actin cytoskeleton. The second mechanism requires an intact F-actin cytoskeleton. Bif seems to be required for this mechanism. Overexpression of Bif can promote actin polymerization in cultured cells. Since it is also known that F-actin forms a complex with Bif in Drosophila embryonic lysates and that Bif binds directly to F-actin filaments in vitro, it is possible that Bif acts to stabilize actin filaments. In this scenario its absence may cause subtle changes in the F-actin cytoskeleton that may affect its capacity to anchor molecules at the cortex when hom is absent. In contrast, hom can function and is required to anchor Osk in the absence of an intact F-actin cytoskeleton or in the absence of bif function. Only when both mechanisms are disrupted in the oocyte, either through the simultaneous disruption of both hom and bif, or when F-actin is disrupted in the absence of hom, do the osk gene products fail to remain anchored to the posterior cortex (Babu, 2004).
Recent studies showed that Drosophila moesin is essential to link the cortical F-actin to the oocyte cell membrane. When moesin function is compromised, cortical F-actin can detach from the cell membrane and "fall" into the oocyte cytoplasm, and this results in the mislocalization of Osk. The effects of loss of moesin function on the localization of both Bif and Hom were examined; in these mutant oocytes, where the cortical F-actin detaches from the mutant oocyte cell membrane, components of both of the proposed anchoring pathways, Hom and Bif, also detach. These observations are consistent both with the Osk mislocalization phenotype seen in moesin mutant oocytes and with the model. It will be interesting to identify additional molecules involved in these separate pathways and to elucidate the mechanisms that are required to localize Hom to the posterior cortex of the oocyte in the absence of an intact actin cytoskeleton (Babu, 2004).
Bifocal is a putative cytoskeletal regulator and a Protein phosphatase-1 (PP1) interacting protein that mediates normal photoreceptor morphology in Drosophila. Bif and PP1-87B, as well as their ability to interact with each other, are required for photoreceptor growth cone targeting in the larval visual system. Single mutants for bif or PP1-87B show defects in axonal projections in which the axons of the outer photoreceptors bypass the lamina, where they normally terminate. The functions of bif and PP1-87B in either stabilizing R-cell morphology (for Bif) or regulating the cell cycle (for PP1-87B) can be uncoupled from their function in visual axon targeting. Interestingly, the axon targeting phenotypes are observed in both PP1-87B mutants and PP1-87B overexpression studies, suggesting that an optimal PP1 activity may be required for normal axon targeting. bif mutants also display strong genetic interactions with receptor tyrosine phosphatases, dptp10d and dptp69d, and biochemical studies demonstrate that Bif interacts directly with F-actin in vitro. It is proposed that, as a downstream component of axon signaling pathways, Bif regulates PP1 activity, and both proteins influence cytoskeleton dynamics in the growth cone of R cells to allow proper axon targeting (Babu, 2005).
Rescue experiments utilizing the two different isoforms of bif demonstrate that the function of Bif in photoreceptor axon guidance can be uncoupled from its function in rhabdomere morphology in photoreceptor cells, and this indicates that Bif has a dual role in the fly visual system, normal axon connectivity in the larval stages and the formation of normal rhabdomeres in the adult eye. It should however be noted that the rhabdomere rescue obtained using both the bif+ and the bif10Da isoforms of bif was not complete, possibly because the expression pattern and/or level of Bif expression in the rescue experiments are somehow different from the wild type expression of Bif. The larger Bif+ isoform is 1196 amino acids long, whereas the shorter Bif10DA isoform is 1063 and is formed by an alternative splice site between exon 4 and 5. It is somewhat surprising to find that the two isoforms of Bif behave differently with respect to axon guidance since both isoforms contain the PP1 and actin-binding sites. One possible explanation is that the additional C-terminal sequences present in Bif+ are required for its function in axon guidance. Interestingly, this region comprises one of the actin-binding domains. The precise role of these sequences in vivo is not clear at present, but it is possible to envisage roles for them in protein folding or mediating binding to other partners. In this regard, one plausible scenario is that the two Bif isoforms may assume distinct patterns of subcellular localization due to their different C-termini. It is possible for future approaches to address this question by generating specific antibodies that recognize the different isoforms of Bif or expressing tagged versions of the two Bif products (Babu, 2005).
Mutations in the conserved phosphatase-binding motif of Bif, which abolish binding of Bif to PP1-87B, render the protein less effective in axon guidance. Similar effects on Bif activity for these mutations occur in the morphogenesis of rhabdomeres. Another interesting aspect is that Bif can form a complex with the cytoskeletal F-actin both in vivo and in vitro. Changes in the actin cytoskeleton are known to be essential for remodeling of the axon growth cone. These data suggest that, in addition to its inhibitory regulatory effect on the phosphatase activity, Bif might serve as a direct link between PP1-87B and the actin cytoskeleton. Similar observations were reported for neurabins, which are the inhibitors of PP1 in mammals. It has been shown that neurabins bind actin and serve as linkers between the actin cytoskeleton and the plasma membrane at the cadherin-based cell-cell adhesion sites. Neurabin I is highly concentrated at the synapse of developed neurons and in the lamellapodia of growth cones during the development of neurons, suggesting that it could be required for synapse formation or function. Although it does not share any sequence homology with mammalian neurabins, Bif could be a functional homologue of neurabins in the Drosophila larval visual system by associating with PP1-87B and the actin cytoskeleton. Such functions of Bif might consequently affect the phosphorylation status of various actin-binding proteins like myosin II and cofilin, which are known to undergo phosphorylation and dephosphorylation (Babu, 2005).
A role for PP1 itself in axonal connectivity in the larval optic lobe is supported by two observations: (1) expressing PP1 inhibitors in post-mitotic cells in the eye imaginal disc results in axon defects; (2) transallelic combinations of pp1-87B mutants, which are able to bypass the earlier defects of cell cycle arrest that are usually seen in homozygotes of strong pp1-87B mutants, reveal axon targeting defects in the larval lamina. Although the PP1 inhibitors are not specific to PP1-87B, they nevertheless do support the notion that the phosphatase activity of PP1 is required for proper axon guidance. In the transallelic combinations of pp1 mutations, formation of the R cells of the larval eye disc is not adversely affected, suggesting that the axon guidance phenotype of pp1-87B mutants is not simply due to the lack of cells in the developing eye disc. Interestingly, both pp1-87B mutants as well as PP1 overexpression give rise to axons bypassing the lamina and entering the medulla of the optic lobe. This suggests that an optimal level of PP1 activity in the eye disc and optic lobe may be required, and either increase or decrease of PP1 levels could lead to changes in the phosphorylation status of target proteins and subsequent defects in the growth cone cytoskeleton and axon guidance (Babu, 2005).
PP1 is one of the most abundant eukaryotic protein phosphatases that dephosphorylate serine and threonine residues of target proteins. The activities of various PP1 catalytic (PP1c) subunits are extensively regulated in various organisms and tissue types within a single organism. PP1c binds various regulatory molecules, which could bring the phosphatase to its site of action or act as adaptors for the phosphatase at its site of function. There are 4 related PP1c subunits in Drosophila. Drosophila PP1s are very similar proteins encoded by different loci in the fly genome, and they are variably regulated at different points in development. It is conceivable that Bif acts as one of the regulatory subunits of PP1 in the fly eye, and it is required either as a complex with PP1 and the actin cytoskeleton or for recruiting PP1 to subcellular sites where the PP1 activity is required to modulate the actin cytoskeleton. The former hypothesis seems more plausible since bif mutant larval and pupal eye discs do not show any obvious changes in PP1 staining (Babu, 2005).
Based on the genetic interactions of bif and the receptor tyrosine phosphatase genes, Dptp10D and Dptp69D, and on the data of Bif functional and biochemical interactions with PP1-87B and F-actin, a possible model for Bif function in axon guidance could be put forward: activation of receptor molecules leads to signaling events (perhaps also involving Misshapen) that activate Bif in the growth cone which in turn leads to inhibition of PP1 activity and changes in the actin cytoskeleton to support axon outgrowth and guidance. In this scenario, PP1 must have a threshold level of activity; decreased levels of PP1-87B and general inhibition of PP1 activity also cause axon guidance defects. Bif might participate in lowering PP1 activity to the threshold level but must not abolish all PP1 activity in order to allow normal photoreceptor axon guidance. Interestingly, overexpression of Bif as four copies has been shown to induce axonal targeting defects (Babu, 2005).
Finally, bif mutants affect axon targeting in the larval optic lobe, but these defects seem to be corrected in the adult. How this axon defect is rectified in the adult stages remains unclear, and it probably occurs during the extensive remodeling that takes place during the pupal stages of fly development. In future studies, it would be interesting to determine if any of the other molecules involved in Drosophila photoreceptor axon guidance behave in a similar manner, and it would perhaps also be informative to classify phenotypes into those that can be repaired and those which cannot (Babu, 2005).
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